1. Field of the Invention
This invention relates to improved methods for preparing poly(ethylene glycol fumarate) and to methods for chemically crosslinking or photocrosslinking poly(ethylene glycol fumarate) with hydrophobic polymers such as poly(propylene fumarate) and poly(caprolactone fumarate) to form various hydrogels with controllable hydrophilicity. The hydrogels are useful as a biocompatible, bioresorbable, injectable, and in-situ hardening scaffold for tissue engineering applications and for controlled drug release applications.
2. Description of the Related Art
The clinical needs for bone regeneration are diverse, and there are roughly 1,000,000 patients who have skeletal defects each year in the United States that require bone graft procedures to achieve union. These include applications arising from resection of primary and metastatic tumors, bone loss after skeletal trauma, primary and revision total joint arthroplasty with bone deficiency, spinal arthrodesis, and trabecular voids following osteoporotic insufficiency fractures.
Current clinical methods of treating skeletal defects involve bone transplantation or the use of other materials to restore continuity. Autologous bone graft has been the gold standard of bone replacement because it provides such essential elements as osteogenic cells, osteoinductive factors, and an osteoconductive matrix for healing. However, the limited supply of autograft bone, and donor site morbidity both restrict the spectrum of cases in which it can be used alone. Allograft bone, although available in abundant supply, has drawbacks that include reduced rates of graft incorporation compared to autograft bone, and the possibility of pathogen transfer from donor to host.
Metals provide immediate mechanical support at the defect site but exhibit less than ideal overall integration with host tissue and can eventually fail due to fatigue loading if the bone does not heal prior to fatigue failure of the metal. Ceramics, such as β-tricalcium phosphate (β-TCP) and hydroxyapatite are both osteoconductive, and have found clinical use as surface coatings on metal prostheses to enhance bonding of those prostheses to bone. In particulate form, they offer increased mechanical strength to polymeric composite materials primarily in compression, but are less effective in enhancing resistance to torsional and bending forces. Poly(methyl methacrylate) bone cement can be injected or molded and is sometimes used to fill both cavitary and segmental defects, such as those that result from the curettage of a giant cell tumor or from the resection of a vertebral body in metastatic disease to the spine, respectively. However, the temperature can rise up to 100° C. during the exothermic polymerization reaction, and the heat released risks local tissue injury. Additionally, poly(methyl methacrylate) is non-biodegradable and can thus accumulate fatigue damage with time and eventually undergo mechanical failure.
Synthetic biodegradable polymers may provide treatment options not currently available. These materials can be manufactured in virtually unlimited supply and the flexibility in their design allows the synthesis of a wide range of polymers with varying mechanical, biologic, degradation, and rheologic properties. For instance, their mechanical and degradation properties can be manipulated by changing the polymer molecular weight during synthesis, and can thus be tailored to fit a particular application. The injectable nature of the skeletal regeneration biomaterial would be ideal to fill defects with limited accessibility or irregular shape. For example, minimally invasive endoscopic techniques now in clinical use would allow the injectable form of the biomaterial to be inserted for posterolateral intertransverse process spinal fusion. This would decrease the surgical trauma from the extensive exposure and muscle stripping that must now be done to put the graft material into position. The injectable material could be placed into cancellous voids from periarticular fractures, osteoporotic spinal fractures, or bone cysts without creating a large access hole in the surrounding cortical bone. These clinical situations represent the motivation for the development of injectable biodegradable polymeric materials for bone tissue engineering.
Controlled release of bioactive molecules such as drugs and growth factors has also become an important aspect of tissue engineering because it allows modulation of cellular function and tissue formation at the afflicted site. The encapsulation of drugs, proteins and other bioactive agents within biodegradable materials is an effective way to control the release profile of the contained substance.
Recently developed injectable materials and hydrogels have fulfilled many design criteria for these diverse medical applications. A polyethylene glycol (PEG) derivative, poly(ethylene glycol fumarate) (PEGF), has been developed as an injectable in-situ crosslinkable and biodegradable hydrogel (see Jo, Macromolecules 2001, 34, 2839; U.S. Pat. No. 6,884,778; and U.S. Patent Application Publication No. 2002/0028189). PEGF is a hydrophilic oligomer of PEG with fumarate moieties synthesized by condensation polymerization of polyethylene glycol with fumaryl chloride. The fumarate groups in this macromer allow for crosslinking in-situ as well as degradation via hydrolysis. A chemical initiation system consisting of ammonium persulfate and ascorbic acid is used to form hydrogels without the need for ultraviolet light (see Temenoff, J. Biomed. Mater. Res. 2001, 59, 429). The attachment of marrow stromal cells (MSCs) on PEGF hydrogel has been investigated with a model cell adhesion specific peptide (see Shin, J. Biomed. Mater. Res. 2002, 61, 169). The model RGD peptide was incorporated into PEGF hydrogel after being coupled to acrylated PEG of molecular weight 3400 g·mol−1 (see Jo et al., “Modification of Oligo(poly(ethylene glycol) fumarate) Macromer with a GRGD Peptide for the Preparation of Functionalized Polymer Networks”, Biomacromolecules 2001, 2, 255).
By altering the PEG chain length of PEGF, the crosslink density, or the initial peptide concentration, hydrogels with a wide variety of physical properties can be synthesized. As the peptide concentration is increased the attachment of MSCs to PEGF hydrogels with PEG molecular weights of 930 and 2860 g mol−1 increased. However, the number of attached MSCs to a PEGF hydrogel of PEG molecular weight of 6090 g mol−1 remained constant regardless of the peptide density. The length of PEG chain in PEGF also influenced the degree of cell attachment. For example, when 1 mmol peptide/g of PEGF hydrogel was incorporated into the PEGF, the degree of cell attachment relative to initial seeding density was 93.9±5.9%, 64.7±8.2%, and 9.3±6.6% for PEGF with PEG molecular weights of 930, 2860, and 6090 g mol−1, respectively. On the other hand, the crosslinking density of the PEGF hydrogel did not significantly affect cell attachment. The interaction was sequence specific because MSC attachment to a RGD modified hydrogel was competitively inhibited when cells were incubated in the presence of soluble RGD prior to cell seeding. These results indicate that altering the peptide concentration can modulate cell attachment to a PEGF hydrogel. PEGF macromer has also been crosslinked with N,N′-methylene bisacrylamide (MBA) to fabricate injectable scaffolds which crosslink in-situ as a cell carrier for mesenchymal stem cells (see Jabbari, 14th Int Symp. Microencap. Proceed. 2000, 54). This system is potentially useful for treatment of osteochondoral defects. A novel combination of redox initiators consisting of ammonium persulfate and N,N,N′,N′-tetramethylethylenediamine (TMED) was used in this system to obtain a neutral pH. Mesenchymal stem cells (MSCs) were successfully seeded in this injectable system. The encapsulated MSCs cultured in complete osteogenic media showed alkaline phosphatase activity and increase in mineralized matrix for up to 21 days.
Poly(propylene fumarate) (PPF) is an unsaturated linear polyester that can be modified or crosslinked through its fumarate double bonds. See, for example, U.S. Pat. No. 5,733,951. Poly(ε-caprolactone) (PCL) is a well-known biodegradable polymer and FDA-approved for use as resorbable sutures. It has excellent biocompatibility and flexibility. PCL was recently studied as a potential material for a temporary joint spacer (see Elfick, Biomaterials, 2002, 23, 4463-7) and tissue engineered skin (see Ng, Tissue Engineering, 2001, 7, 441-55). There has been developed a copolymer based on PCL and fumarate segments, poly(caprolactone fumarate) (PCLF). Due to the presence of PCL unit, the PCLF chain is much more flexible than the PPF chain. This renders PCLF self-crosslinkable without the use of any crosslinkers. See PCT International Publication No. WO 2005/004811.
Photocrosslinking is the formation of a covalent linkage between two macromolecules or between two different parts of one macromolecule. Photocrosslinking allows in vivo curing, which provides great flexibility for the placement and handling of implantable polymers for surgeons. The main advantages of photocrosslinking over other crosslinking techniques are spatial and temporal control of the polymerization, fast curing rates at room temperature, and ease of fashioning and flexibility during implantation (see Anseth, Nature Biotechnology, 1999, 17, 156-9).
The major shortcomings of previous poly(ethylene glycol fumarate) (PEGF) synthesis methods are the dark color of the PEGF product and the relatively low efficiency of reaction due to the proton scavenger triethylamine in the polycondensation.
Accordingly, there is a need for improved methods for preparing poly(ethylene glycol fumarate). Also, there is a need for methods for chemically crosslinking or photocrosslinking poly(ethylene glycol fumarate) with hydrophobic polymers such as poly(propylene fumarate) (PPF) and poly(caprolactone fumarate) (PCLF) to form various hydrogels with controllable hydrophilicity as well as controlled swelling and mechanical properties.